High fidelity of internal strand transfer catalyzed by human immunodeficiency virus reverse transcriptase.
Abstract
A system to study the fidelity of internal strand transfer events was constructed. A donor RNA, on which reverse transcriptase (RT)-directed DNA synthesis was initiated, shared homology with an acceptor RNA, to which DNAs initiated on the donor could transfer. The homology occurred over a 119-base internal region of the donor which coded for the N-terminal portion of the alpha-lac gene. Polymerase chain reaction (PCR) was used to amplify DNA synthesis products. The PCR products were then digested with PvuII and EcoRI and ligated into a vector which had this same region excised. Transformed Escherichia coli were screened for the ability to produce a functional beta-galactosidase protein by blue-white phenotype analysis with white colonies scored as those with errors in alpha-lac. Products synthesized on the donor were used to assess the error rate of human immunodeficiency virus-RT while products transferring to and subsequently extended on the acceptor (transfer products) were used to monitor transfer fidelity. Human immunodeficiency virus-RT made approximately 1 error per 7500 bases copied in the assay. Nucleocapsid protein (NCp), although stimulating strand transfer 3-fold, had no effect on RT fidelity. Transfer products in t...Continue Reading
References
Citations
Structures of complexes formed by HIV-1 reverse transcriptase at a termination site of DNA synthesis
Related Concepts
Related Feeds
ASBMB Publications
The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.